Natural mutations in humans and introduced mutations in mice have unequivocally demonstrated the importance of interferon-gamma (IFN-gamma) for the control of tuberculosis (TB). How this cytokine exerts its protective effects is thought to owe much to the broad transcriptional programs it activates within Mycobacterium tuberculosis (Mto)-infected macrophages and dendritic cells. Here as many as 1,300 genes may be engaged. Prominent within this group is a new family of 47kDa guanosine 5'-triphosphatases (p47 GTPases) that confers host resistance to a number of human pathogens. At least one member of this family - LRG-47 - appears essential for combating TB in experimental mouse models. Preliminary evidence suggests that LRG-47 operates at the level of the infected cell via a mechanism distinct from all known tuberculocidal pathways, linking cytokine activation with phagolysomal fusion, events that are required for bacterial killing and which may enable Mtb antigens to be cross-presented. It is the purpose of this proposal to build on these initial observations by pursuing the following aims: (1) Characterize the intracellular location and trafficking behavior of mouse and human LRG-47 within Mtb-infected cells. Here a combination of high-resolution imaging and cell fractionation will be used to identify the LRG-47-positive compartment(s) and its recruitment to the nascent Mtb phagosome. (2) Define the molecular determinants of LRG-47 function in response to Mtb. Relocation of LRG-47 to the Mtb phagosome and subsequent remodeling of this organelle is likely to enlist other host proteins. Isolating LRG-47-interacting partners, the functional domains involved and their consequences for immunity will be dissected with an array of molecular, cellular and structural approaches. (3) Uncover Mtb-encoded pathways that counter LRG-47 - dependent immunity. Genetic screens conducted to identify bacterial components interfering with LRG-47 should yield further insights into how this GTPase operates. It could also uncover prospective drug targets. Information gleaned from these approaches will provide a paradigm for other members of the p47 GTPase family that is rapidly emerging as one of the most powerful host defense repertoires in the mammalian genome.